Derivatives of magainin and methods of production thereof

ABSTRACT

The present invention provides a Magainin derivative peptide and method of production thereof. Also provided is a pharmaceutically composition comprising said Magainin derivative peptide and pharmaceutically acceptable carrier and/or pharmaceutically compatible binding agents. The Magainin derivative peptide of the present invention having amino acid sequence of the general formula shown as below: 
 
Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-X-Asn-Y-Z-OH 
in which: X is an amino acid residue selected from the group consisting of Met, Ile and Leu; Y is an amino acid residue selected from the group consisting of Ser, Lys, Ile, Arg and Leu; and Z is Arg.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of PCT/CN02/00317, whichbears an international filing date of May 8, 2002 and claims priority toChinese Application No. 01112855.0 filed May 10, 2001.

BACKGROUND OF THE INVENTION

There are many life forms, for example, insects, microorganisms,amphibians and human beings which may produce anti-bacterial peptidematerials that protect their communities. These anti-bacterial peptidescan penetrate lipids on the cell membranes and make them inactive, canalso affect protozoon species, germ cells and even viruses, hence suchpeptides are referred to as super-antibiotics. Anti-bacterial peptidesall carry various amounts of positive charge, and their anti-bacterialmechanism lies in the combination (of the positive charges carried bythe peptides with the negative charges carried by the phospholipidswhich exist in the bacteria cell wall, creating an ion path on the cellmembrane, enhancing the penetrability, causing the bacteria to dissolveand die. Hence the anti-microbial activities of these peptides do notdepend on the binding with any specific receptors.

The anti-bacterial peptides exhibit a broad spectrum of antimicrobialactivity upon gram-positive and gram-negative bacteria, as well asaerobic and anaerobic bacteria. They are different from antibiotics inthat anti-bacterial peptides do not produce drug-resistance effects,even bacteria that have resistance to many types of antibiotics could besuppressed by the anti-bacterial peptides. Further, such anti-bacterialpeptides also have inhibitive effect to protozoon species and viruses.As the metabolism products of the anti-bacterial peptides are aminoacids, these peptides are of low toxicity for host cells. In summary,the antimicrobial peptides are a class of compounds with wide prospectsof being used for anti-microbial drugs.

Magainins are a category of naturally occurring anti-bacterial peptidesderived from frog skin with anti-bacterial effects. Magainins has beenextensively studied up till now, they have such features as being easyto be synthesized, low in cost and little possibility of hemolysis.

U.S. Pat. No. 5,589,364 disclosed a method by which the Magainin IIpeptide having 23 amino acids can be prepared using bioengineeringtechniques. Magainin II (hereinafter referred as naturally occurringMagainin, wild-type Magainin or Magainin) is a type of the naturallyoccurring frog-skin anti-bacterial peptide, having amino acid sequenceshown as below:Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Asn-Ser-OH

in which:

Gly stands for glycine, Ile for isoleucine, Lys for lysine, Phe forphenylalanine, Leu for leucine, His for histidine, Ser for serine, Alafor alanine, Val for valine, Glu for glutamic acid, Met for methionineand Asn for asparaginate.

U.S. Pat. No. 6,183,992 disclosed a method to produce MSI-78 (22 aminoacids), a derivative of magainin, having the amino acid sequence shownas below:Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys-NH₂

It has been reported in ADIS NEW DRUG PROFILE by Harriet M. Lamb, etc.,that the Magainin derivative MSI-78 shows obvious curative effect intreating trauma infection and crura ulceration caused by the diabetesmellitus.

BRIEF DESCRIPTION OF THE INVENTION

One aspect of the present invention provides Magainin derivativepeptides having the property of anti-microbial effect.

In one embodiment, the invention is directed to Magainin derivativepeptides in which Arg is added to the C-terminus of the naturallyoccurring Magainin for a cleavage reagent while preserving theanti-microbial activity of the Magainin peptide. In this embodiment, thecleavage reagent includes alkaline protease such as trypsin andclotrispain.

In another embodiment, the present invention is directed to fusionpeptides comprising at least two tandemly linked Magainin derivativepeptides that are altered preferably by adding Arg at the C-terminus ofthe peptides while preserving the anti-microbial activity, as well asisolated DNA sequences comprising a DNA sequence encoding thesepeptides, expression vectors comprising these isolated DNA sequences,and transformed host cells comprising these expression vectors.

Another aspect of the present invention provides the recombinant andsynthetic methods of producing these Magainin derivatives.

In one embodiment, the present invention includes a method for producingthe Magainin derivative peptide of claim 1, either by expressing asingle copy of the Magainin derivative in an expression vector or,alternatively, by expressing a fusion protein containing multiple copiesof the Magainin derivative and then cleaving this fusion protein intoindividual copies of the Magainin sequence using the appropriatecleavage reagent. Cleavage reagents include, but are not limited to,alkaline proteases such as trypsin and clotrispain.

In another embodiment, the present invention includes a method forproducing the Magainin derivative peptide of claim 1 by solid phasesynthesis, which comprises of using HMP resin as a solid phase carrier,protecting alpha-amine of an amino acid with 9-fluorenyl methoxycarbonyl(Fmoc), synthesizing on a peptide synthesizer, and obtaining the peptideafter the steps of separation, purification and lyophilization.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising the Magainin derivative peptide of claim 1 andpharmaceutically acceptable carrier and/or pharmaceutically compatiblebinding agents.

In yet another aspect, the present invention provides a method for thetreatment of a patient having need of the Magainin derivative peptide ofclaim 1 comprising: administrating to said patient a therapeuticallyeffective amount of the peptide of claim 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the process used to construct a plasmid containing 1 to16 copies of the gene encoding the Magainin derivative peptide.

FIG. 2 shows the result of the time-kill study of Magainin (Leu₂₁,Ser₂₃, Arg₂₄) on Escherichia coli.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel Magainin derivatives withanti-microbial properties, and to recombinant and synthetic methods forproducing these Magainin derivatives. The recombinant methods encompassboth methods directed to single copies of the genes for these peptides,and, preferably, to multiple copies of these genes that are tandemlylinked so as to produce fusion proteins which are then cleaved toproduce multiple copies of the desired peptide.

In the latter case, one embodiment of the present invention isspecifically directed to the alteration of the sequences of thesepeptides to allow for the cleavage of the multimeric fusion proteinsproduced by these methods by trypsin, which normally is specific foreither Arg or Lys residues. In the methods of the invention, Arg ispreferably added to the C-terminus of each gene, and any Lys residuesinternal to these genes are reversible protected to prevent suchresidues from trypsin cleavage.

The present invention is also directed to various pharmaceuticalformulations containing the Magainin derivatives of the presentinvention. Formulations containing these active compounds havetherapeutic utility, particularly in the treatment of disease caused bymicrobial infection.

In the present invention, “anti-microbial” peptides are peptides withMagainin-like anti-microbial activity, i.e., peptides that haveinhibitive property to bacterial reproduction or proliferate. Assays forsuch activity are well known to the skilled artisan, and are describedelsewhere herein (see, e.g., the anti-microbial assay models provided inthe examples). Anti-microbial peptides contemplated herein includeMagainin, and analogs and derivatives thereof, including the specificanalogs arid derivatives disclosed elsewhere heroin. As intended herein,“derivatives,” “analogs,” and “Variants” are used synonymously.

Sequences of Magainin Derivatives

The present invention is directed to derivatives of the naturallyoccurring Magainin, which has the amino acid sequence as below:Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Asn-Ser-OH

In one aspect of the invention, the derivatives of Magainin contemplatedhave amino acid sequences of the general formula as below:Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-X-Asn-Y-Z-OH

In which:

X is an amino acid residue selected from the group consisting of Met,Ile and Leu; Y is an amino acid residue selected from the groupconsisting of Ser, Lys, Ile, Leu and Arg, and; Z is Arg.

Of particular interest are the Magainin derivatives of SEQ ID NOs:1-15,which are shown elsewhere herein to exhibit particularly advantageousproperties. As set forth in the present invention, the Magaininderivatives contemplated herein may be prepared by synthetic chemicalmeans, and by less expensive recombinant techniques that are novel tothe present invention.

In the present invention, Magainin derivatives are generally referred toeither by SEQ ID NO, or, alternatively, by following the name “Magainin”with a designation within parentheses of each changed a o acid positionin the sequence, where the new amino acid(s) at each changed positionis/are given, followed by a subscript indicating the position of thechange(s) relative to the N-terminal amino acid of the peptide. Thus, inthis alternative nomenclature, SEQ ID NO:1 may also be designated asMagainin (Met₂₁, Ser₂₃, Arg₂₄).

Other Magainin derivatives contemplated herein include, e.g.: Magainin(Met₂₁, Lys₂₃, Arg₂₄) (SEQ ID NO: 2); Magainin (Met₂₁, Arg₂₃, Arg₂₄)(SEQ ID NO: 3); Magainin (Met₂₁, Ile₂₃, Arg₂₄) (SEQ ID NO: 4); Magainin(Met₂₁, Leu₂₃, Arg₂₄) (SEQ ID NO: 5); Magainin (Ile₂₁, Ser₂₃, Arg₂₄)(SEQ ID NO: 6); Magainin (Ile₂₁, Lys₂₃, Arg₂₄) (SEQ ID NO: 7); Magainin(Ile₂₁, Ile₂₃, Arg₂₄) (SEQ ID NO: 8); Magainin (Ile₂₁, Arg₂₃, Arg₂₄)(SEQ ID NO: 9); Magainin (Ile₂₁, Leu₂₃, Arg₂₄) (SEQ ID NO: 10); Magainin(Leu₂₁, Ser₂₃, Arg₂₄) (SEQ ID NO: 11); Magainin (Leu₂₁, Lys₂₃, Arg₂₄)(SEQ ID NO: 12); Magainin (Leu₂₁, Ile₂₃, Arg₂₄) (SEQ ID NO: 13);Magainin (Leu₂₁, Arg₂₃, Arg₂₄) (SEQ ID NO: 14) and Magainin (Leu₂₁,Leu₂₃, Arg₂₄) (SEQ ID NO: 15).

The Magainin derivatives of this invention are amphoteric (synonymouswith amphiprotic) compounds, and may be sufficiently acidic orsufficiently basic to react with any of a number of inorganic bases, andinorganic and organic acids, to form a salt. Acids commonly employed toform acid-addition salts are inorganic acids such as hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, aidthe like, and organic acids such as p-toluenesulfonic acid,methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, acetic acid, and thelike. Examples of such salts include the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like. Preferred acidaddition salts are those formed with mineral acids such as hydrochloricacid and hydrobromic acid, and, especially, hydrochloric acid.

Alkalis may also be employed to react with derivatives of this inventionto form salts. Representative examples of such alkalis include ammonium,alkali metals, alkali metal hydroxides, carbonates, and bicarbonates.Typically, such an alkali may be sodium hydroxide, potassium hydroxide,ammonium hydroxide, and potassium carbonate.

One aspect of the present invention provides a method to produce anMagainin derivative peptide thereof by solid phase synthesis, whichcomprises of using HMP resin as a solid phase carrier, protecting thealpha-amine of an amino acid with 9-fluorenyl methoxycarbonyl (Fmoc),synthesizing the peptide on a peptide synthesizer by the amino acidsequence of an Magainin derivative, and obtaining the peptide afterseparation, purification and lyophilization. For example, all theMagainin derivative peptides of the present invention may be synthesizedby such chemical methods.

In another aspect, the present invention provides a method to produce aMagainin derivative peptide by recombinant techniques. In oneembodiment, such techniques comprise: synthesizing gene fragments by theamino acid sequence of an Magainin derivative; ligating the synthesizedgene fragments; constructing a recombinant plasmid, culturing suitablebacterial host cells and transforming the recombinant plasmid into thebacterial host cells; extracting the inclusion bodies after fermentationof the bacteria strain and collapse of cell walls; and, obtaining thefinal product after lysing the inclusion bodies, separating roughproduct followed by HPLC purification and lyophilization.

Although the present invention discusses the construction of the desiredMagainin derivative gene of interest via synthetically produced DNAfragments, also contemplated herein are any suitable method forproducing the gene of interest e.g., by mutagenesis of the wild-typeMagainin gene, or any other method known to the skilled artisan.

In an additional embodiment, the present invention contemplates arecombinant method comprising: constructing an expression vectorcomprising at least two tandemly linked Magainin derivative DNAsequences and a promoter sequence, wherein the promoter sequence iscapable of driving the expression of the tandemly linked Magaininderivatives DNA sequences; expressing the expression vector in a hostcell to produce a fusion protein comprising at least two tandemly linkedMagainin derivative peptide sequences; and, cleaving the resultingfusion protein into separate Magainin derivative peptides.

In one aspect of the present invention, recombinant methods may be usedto produce monomers of the Magainin peptide derivatives of interest,e.g., monomers of the Magainin peptide derivatives of the invention. Inanother aspect, the present invention is drawn to the production byrecombinant methods of fusion proteins of the Magainin peptidederivatives of interest, e.g., multimeric fusion proteins of theMagainin peptide derivatives of the invention.

In such tandemly linked Magainin or Magainin derivative constructs,“tandemly linked” or “tandem linkage” as it refers to the peptides ofthe invention is used to indicate any linkage between the peptides ofinterest that allows for the production of a single fusion protein thatmay be cleaved by the appropriate cleavage reagent to produce separatepeptides of the desired sequence. As described below in the discussionof Magainin, such cleavage reagents include, e.g., alkaline proteasessuch as trypsin and clotrispain.

In the present invention “tandemly linked” or “tandem linkage” is alsoused to refer to the joining of DNA sequences of the invention. In thiscontext, “tandemly linked” DNA sequences are DNA sequences that are sojoined that they serve as the template for the production bytranscription/translation of a tandemly linked fusion peptide.

In one embodiment of the present invention, two or more Magaininderivative sequences may be tandemly linked without a spacer sequence.As an example, two Magainin derivative sequences in which an Arg residuehas been added to the C-terminus (e.g., any of SEQ ID NOs 1-5) may betandemly linked such that the Arg at the C-terminus of the firstMagainin derivative sequence is followed by the Gly at the N-terminus ofthe next Magainin derivative sequence. Similar tandem linkage of morecopies of such Magainin derivative sequences allow for the production ofa fusion protein in which N copies of the Magainin sequence arerepeated. In these constructs, treatment with, e.g., trypsin, willcleave the fusion protein after each C-terminal Arg residue to produceseparate Magainin derivative sequences.

In another embodiment, Magainin derivative sequences may be tandemlylinked with an amino acid spacer of Xaa . . . Xaa between the C-terminusof one peptide and the N-terminus of the next peptide in the fusionprotein. In this circumstance, in order for the fusion proteinscontaining tandemly linked Magainin derivative sequences to be correctlycleaved to produce separate peptides of the desired sequence, it isnecessary that cleavage does not occur within these sequences, while thespacer amino acid Xaa . . . Xaa is cleaved from linking with theC-terminus or the N-terminus of any desired separate peptide.

With regard to using trypsin as a cleavage reagent, any of the internalLys₄, Lys₁₀, Lys₁₁ and Lys₁₄ amino acid residues of the wild-typeMagainin sequence that are present in the fusion protein should beprotected from being cleaved. Thus in instances where the cleavagereagent has an internal recognition site or sites within the Magaininderivatives of the fusion protein, it will be necessary to protect theinternal Lys within these sequences. For example, acetylation methods asdescribed elsewhere herein may be used to protect internal Lys residues,thereby removing them after trypsin cleavage.

In making substitutions to the Magainin sequence, it is important tomaintain the activity of the Magainin derivatives produced by suchsubstitutions. Such conservation of activity may be made based onpredictions as to appropriate substitute amino acid residues, forexample based on conservative or highly conservative amino acidsubstitutions. Predictions of appropriate amino acid substitutions mayalso be made based on data regarding sequence conservation at particularamino acid positions.

Alternatively, substitutions that maintain the activity of the Magaininderivatives produced by such substitutions may be identified by thescreening of Magainin derivatives for Magainin-like activity.Specifically, random or directed mutations in the Magainin sequenceproduced by standard means may be screened for their effect on theactivity of the resulting Magainin derivative peptide by activity assaysappropriate for Magainin. In this regard, “Magainin-like activity,” asused herein, refers to anti-microbial activity exhibited by Magainin inany of the assays disclosed elsewhere herein.

As used herein, “derivatives,” “analogs,” and “variants” are usedsynonymously to refer to sequences derived from and related to theMagainin sequence, as described in detail below. Specific examples ofMagainin derivatives contemplated herein include the sequences of SEQ IDNOs:1-15.

In accordance with a preferred embodiment of the present invention,Magainin derivative peptides also include any of the above-describedsequences modified to have at least 85% percent similarity, preferably90% similarity, while preserving Magainin-like activity. For example,derivatives may include sequences in which, in addition to an Arg addedto the C-terminus, have up to three amino acid changes while stillretaining Magainin-like activity. Such small changes in sequence wouldbe well-known to one of ordinary skill in the art. Examples of suchchanges may be found in the relevant literature for Magainin peptides.

Also contemplated as included within the term “Magainin derivatives” areMagainin derivative sequences that are further modified by any of themodifications known to the skilled artisan, particularly thosemodifications that improve the properties of the Magainin derivative,e.g., the half-life of the peptide.

As contemplated herein, “Magainin derivatives” also encompasseswild-type Magainin sequences where modifications are made not to theamino acid sequence itself, but to the side-chains of the amino acids,i.e., by cross-linking of reagents known to one of ordinary skill in theart etc.

Recombinant Method for Producing Magainin Derivatives

The recombinant techniques used to prepare constructs, expressionvectors, transformed cells, and purified fusion proteins of suchMagainin derivatives are described as below. Specific examples of theuse of recombinant techniques to prepare monomeric or multimericpeptides of the Magainin derivatives of the invention are provided inthe Examples.

It is well known that one amino acid may be encoded by multiple codons.Thus a Magainin derivative DNA sequence, as used herein, refers to anyDNA sequence that encodes a specified Magainin derivative peptidesequence. One skilled in the art can deduce and synthesize various DNAsequences and sequence combinations encoding Magainin derivative. In thepresent invention, codons with high frequency in E. Coli are preferred.

Vectors suitable for carrying the DNA sequences encoding Magaininderivative can be chromosome-derived, non-chromosome-derived, orsynthetic DNA. These vectors may include, but are not limited to,microphage DNA, bacillus virus, bacterial plasmid, yeast plasmid, andvectors derived from a combination of phage, plasmid and viral DNA. Theviral DNA may include, but is not limited to, bovine and poultry smallpox virus, adenovirus, and pseudorabies virus. Many other suitablevectors are well known to one skilled in the art. Any plasmid or vectorthat exist and replicates stably in host cells may be used in thisinvention.

Representative but non-limiting examples of the expression vectorscontemplated in the present invention include those used in bacterialsystems, such as commercially available plasmids pKK233-2, pKK223-3,pEZZ18, pUC18, pUC19, and pT7 (Amersham Pharmacia Biotech).

In the present invention the target gene is linked to an appropriatepromoter on an expression vector. A promoter is a sequence that canregulate and control gene transcription, i.e., is capable of driving theexpression of a protein sequence using a DNA template. Therepresentative examples of promoter include lac, trp, tac of E, Coli; T7of phage; P_(L) of λ phage, and other known promoters existing inprokaryotic cells, eukaryotic cells, and viruses that control geneexpression. Particularly preferred bacterial promoters include lacIlacZ, T3, T7, Protein A signal peptide, gpt, λP_(R), P_(L) and trp. Theselection of appropriate promoters is apparent to one skilled in theart.

In addition, the preferred expression vector may have one or moreselection marker gene(s) in order to facilitate screening of the hostcells. Such marker genes include tetracycline and penicillin resistancegenes in E. Coli, and dihydrofolate reductase and neomycin resistancegenes in eukaryotic expression systems.

The expression vectors of the present invention may contain N copies ofthe genes linked in tandem, in which N is an integer from 1 to 16.Preferably, N is an integer from 2 to 8. More preferably, N is either 4or 8. Thus in one preferred embodiment of this invention, the expressionvector contains 1 copy of Magainin derivative. In another preferredembodiment of this invention, the expression vector contains 2 copies ofMagainin derivative linked in tandem. In another preferred embodiment ofexample presented in this invention, the expression vector contains 4copies of Magainin derivative linked in tandem. In another preferredembodiment of this invention, the expression vector contains 8 copies ofMagainin derivative linked in tandem. In another preferred embodiment ofthis invention, the expression vector contains 16 copies of Magaininderivative linked in tandem.

The vectors of the present invention carrying multiple copies of gene(s)and appropriate promoters or other gene expression regulatory componentscan be transformed into appropriate host cells to express the fusionproteins in the host cells. Therefore, this invention also relates tohost cells that are capable of expressing Magainin derivativepolypeptides. The expression vector can be introduced into host cells bygenetic engineering method such as transformation, transfection, orinfection. For example, the expression vector may be introduced viatransformation with calcium chloride, transfection in the presence ofDHAF-dextran as a carrier, or by electroperforation. These methods willefficiently transfer the vector containing multiple copies of gene(s) ofthe present invention into) host cells. The vectors referred to hereincan be plasmids, viral particles, or bacterial phages.

Suitable host cells may include, but are not limited to, bacterial cellssuch as E. Coli, streptococcus, salmonella, and eukaryotic cells such asyeast. The selection of the appropriate host cells is apparent to oneskilled in the art. For the purpose of lowering production cost,prokaryotic cells are the preferred host cells. Representative examplesinclude a variety of strains of E. coli, e.g., JM103, JM109, HB101, andDH5α.

The host cells of the present invention contain an expression vectorcontaining N copies of a gene encoding Magainin derivative peptides, inwhich N is an integer from 1 to 16. Correspondingly, the host cellsexpress fusion proteins containing N copies of Magainin derivativelinked in tandem, in which N is preferably, N is an integer from 2 to 8.More preferably, N is either 4 or 8. The fusion protein does not containany other carrier proteins.

The genetically engineered bacterial strains of the present inventionare cultured under appropriate conditions to produce and accumulatefusion proteins composed of N copies of the linked polypeptides. Theculturing conditions such as culturing media, temperature, humidity andpH value are apparent to one skilled in the art.

After the host cells have grown to a proper density, they can beharvested, e.g., by centrifugation. The harvested cells are thenruptured by physical or chemical methods, and the resulting product iscollected and subject to further purification.

The microorganism cells expressing recombinant proteins can be rupturedby any conventional means, which may include, but are not limited to,freeze and thaw cycles, ultrasonic or mechanical treatment, or cellularlysis reagents. The selection of appropriate protocols to break up hostcells is apparent to one skilled in the art.

The fusion proteins presented in the present invention are composed ofmultiple, Magainin derivative peptides. Under suitable cleavageconditions and with proper substances, the fusion protein may be cleavedat the N-terminus of each Magainin derivative peptide, thereby producingmultiple Magainin derivative peptides.

Since the C-terminus of Magainin derivative is an Arg residue, inanother preferred embodiment of the present invention, the proteasetrypsin was used to specifically cleave the peptide bond formed by theparticipation of the carboxyl group of Arg. In this situation, it may benecessary to alter trypsin reaction conditions to prevent trypsincleavage at internal Lys residues.

Alternatively, various anhydrides may be used in this process to protectany internal Lys residue or residues from cleavage by trypsin. Forexample, the present invention specifically contemplates acetylation ofthe two internal Lys residues of Magainin derivative by aceticanhydride, citraconic anyhdyride, or 3,4,5,6-tretrahydophthaloylanhydride as a superior alternative for the protection of these residuesfrom trypsin cleavage. As a result, trypsin can be used to specificallycleave the peptide bond formed by the participation of the carboxylgroup of Arg without cleaving at any Lys residues. Therefore one step ofcleavage can yield multiple Magainin derivative peptides.

In accordance with a preferred embodiment with the method of the presentinvention, acetylation of the internal Lys₄, Lys₁₀, Lys₁₁ and Lys₁₄residue in Magainin may be accomplished by acetylation of the ε-NH₂ inthe Lys residues. Such acetylation is conducted by, e.g., suspending thepurified wet inclusion body in a Na₂CO₃ solution, and then graduallyadding maleic anhydride derivatives to the solution with stirring atroom temperature at pH 8. After 4 hours, the reaction mixture isdialysed overnight in phosphate buffer, and the fusion protein in thedialyzed reaction mixture is digested with trypsin at a ratio ofprotein-to-trypsin of about 1000:0.5-2 (w/w) at 30° C. for 2 hours.During the reaction, digestion is monitored by HPLC analysis.

After digestion, the acyl group from the ε-NH₂ is deprotected byacidifying the reaction mixture to pH 2.0-3.0 for 4-6 hours at roomtemperature using hydrochloric acid. Finally, the acidified reaction isneutralized with NaHCO₃ to pH 5.0, the precipitate is centrifuged, andthe crude Magainin derivative is collected from the precipitate.

After fusion protein cleavage, highly purified polypeptide can beobtained via a series of separation and purification steps, e.g., bychromatographic methods. Such chromatographic methods may include, butare not limited to, ion-exchange, hydrophobic, size exclusion, andreverse phase chromatography. The media used in these methods may bepurchased from commercial vendors, such as Amersham Pharmacia Biotech,Whatman, Merk KgaA, and Grace Vydac etc. Single chromatography or acombination of multiple Chromatography steps may also be used in thepurification processes. In general, HPLC is used as a means ofpurification. Typically, C18 reversed phase chromatography with aTFA-CH₃CN system as mobile phase is utilized. These chromatographicmethods are well known to one skilled in the art.

It should be pointed out that, although the method to produce Magaininderivative peptide has been described hereinafter to illustrate thepresent invention, it should be apparent to one skilled in the art basedon the disclosure presented herein that such method can also be used toproduce Magainin analogs, as long as the amino acid residue at theN-terminus and at the C-terminus of a Magainin derivative analog canform a specifically cleavable peptide bond with the neighboring aminoacid residue(s), while the cleavage will not occur internally within thepolypeptide. Therefore, methods to produce Magainin analogs by ligatinggenes in tandem are within the scope of the present invention.

With regard to the production of Magainin derivative, the recombinantmethods of the present invention have a number of advantages over othermethods. Chemical synthesis of Magainin derivative, for example, istechnically demanding, and the cost of such synthesis is high.

Pharmaceutical Compositions

The Magainin derivatives of the present invention can be incorporatedinto pharmaceutical compositions. Such compositions typically includethe Magainin derivatives of the present invention (synonymously, “activecompound”) and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose, pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an anent which delaysabsorption for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such ads ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral, buccal, parenteral or inhalationcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and thereby reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein or polypeptide can include a single treatment or,preferably, can include a series of treatments.

The present invention is further illustrated by the following examples,which should not be construed as limiting, but are merely exemplary innature. The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference.

EXAMPLES Example 1 Preparation of the Magainin (Leu₂₁, Ser₂₃, Arg₂₄) byBioengineering Techniques

I. Construction of a Plasmid Containing One Copy of the Gene EncodingMagainin (Leu₂₁, Ser₂₃, Arg₂₄)

A. Synthesis of Gene Fragments

The following gene fragments were synthesized by the amino acid sequenceof (Leu₂₁, Ser₂₃, Arg₂₄) in Shanghai Genecore Biotechnologies Co., Ltd.F₁: 5′ AAT TCC AGA TCT CGT ATG GGT ATC GGT AAA TTT CTG CAC AGC GCG AAAAAA 3′ F₂: 5′ TTT GGT AAA GCG TTT GTG GGT GAA ATC CTG AAC AGC CGT TAGGGA TCC A 3′ F₃: 5′ AG CTT GGA TCC CTA ACG GCT GTT CAG GAT TTC ACC CACAAA CGC TTT 3′ F₄: 5′ ACC AAA TTT TTT CGC GCT GTG CAG AAA TTT ACC GATACC CAT ACG AGA TCT GG 3′

B Litigation of DNA Fragments

The systemized fragment F₁, F₂, F₃ and F₄ with optical density at 260 nm(A_(260nm)) equaling to 2 were dissolved in 40 μl sterile water,respectively. 2.5 μl of fragment F₁ and F₄ were drawn into one tubesseparately, while 2.5 μl of fragment F₂ and F₃ were put into in anothertube separately. To the two tubes, 1 μl of T₄ polynucleotide kinase, 1μl of 10×T₄ polynucleotide kinase buffer, 0.5 μl of ATP with aconcentration of 0.1 mol/L, and 2.5 μl of sterile water were addedrespectively.

The reaction mixture was incubated at 37° centigrade for 60 minutes,then incubated in a water bath at 95° centigrade for 10 minutes to makeT4 kinase inactives, and then was naturally cooled down to roomtemperature to complete the anneal of complementary DNA fragments.

1 μl of T₄ Ligase and 2 μl of the T₄ Ligase buffer were added. Themixture was incubated overnight at 15° centigrade for litigation.

The contemplated DNA fragments after litigation could encode Magainin(Leu₂₁, Ser₂₃, Arg₂₄) having amino acid sequence shown as below: EcoRI Bgl II   Asn Ser Arg Ser Arg Met Gly Ile Gly Lys Phe Leu His SerAla Lys Lys Phe Gly Lys

Ala Phe Val Gly Glu Ile Leu Asn Ser Arg stop Gly Ser

The plasmid pUC8 was double digested with EcoRI and Hind III, 10 μl(about 1 μg) of pUC8, 0.5 μl of EcoRI, 0.5 μl of Hind III, 2.0 μl ofmuticore buffer, 0.2 μl of BSA, and 6.8 μl sterile water were added intoa tube, and the reaction mixture was incubate at 37° centigrade for 2hours. The digested vector was purified and recovered by electrophoresistough low melting point agrose, then precipitated by alcohol, anddissolved in 10 μl of sterile water.

0.5 μl of the digested plasmid pUC8 was ligated with the DNA fragmentobtained from step B, the ligation reaction was conducted at 16°centigrade for 5 hours.

The bacterial cells of E. coli JM103 JM103, JM 109 or DH5α was culturedand treated with calcium chloride for preparing the competent bacterialcells, according to the procedures described in Molecular Cloning: aLaboratory Manual, published by Cold Spring Harbor Laboratory Press. Theproduct resulted from ligation of the digested plasmid and the DNAfragment was added into 100 μl competent bacterial cells fortransformation. The transformed bacterial cells were incubated on icefor 30 minuets, then incubated at 42° centigrade for 2 minuets, andagain incubated on ice for 2 minuets. The bacterial cells were addedinto 300 μl of LB liquid media, followed by incubation at 37° centigradefor 1 hour. Bacterial cells were spread on a plate containing LB mediaand ampicillin antibiotic, and incubated overnight at 37° centigrade.Single colonies were screened in the following day, from which therecombinant plasmid was extracted. The DNA sequencing of the recombinantplasmid was performed on a sequence analyzer. The recombinant plasmidcontains two copies of the gene encoding Magainin (Leu₂₁, Ser₂₃, Arg₂₄)is referred as pUC-M1.

II. Construction of a Plasmid Containing Multiple Copies of the GeneEncoding Magainin (Leu₂₁, Ser₂₃, Arg₂₄)

Synthesis of Gene Fragments

The following gene fragments were synthesized by the amino acid sequenceof (Leu₂₁, Ser₂₃, Arg₂₄) in Shanghai Genecore Biotechnologies Co., Ltd.F₁′: 5′-AAT TCC AGA TCT CGT GGT ATC GGT AAA TTT CTG CAC AGC GCG AAAAAA-3′ F₂′: 5′-TTT GGT AAA GCG TTT GTG GGT GAA ATC CTG AAC AGC CGT GGATCC TAG A-3′ F₃′: 5′-AG CTT CTA GGA TCC ACG GCT GTT CAG GAT TTC ACC CACAAA CGC TTT-3′ F₄′: 5′-ACC AAA TTT TTT CGC GCT GTG CAG AAA TTT ACC GATACC ACG AGA TCT GG-3′

B. Litigation of DNA Fragments

The systemized fragment F₁′, F₂′, F₃′ and F₄′ with optical density at260 nm (A_(260nm)) equaling to 2 were dissolved in 40 μl sterile water,respectively. 2.5 μl of fragment F₁′ and F₄′ were drawn into one tubesseparately, while 2.5 μl of fragment F₂′ and F₃′ were put into inanother tube separately. To the two tubes, 1 μl of T₄ polynucleotide,kinase, 1 μl of 10×T₄ polynucleotide kinase buffer, 0.5 μl of ATP with aconcentration of 0.1 mol/L, and 2.5 μl of sterile water were addedrespectively.

The reaction mixture was incubated at 37° centigrade for 60 minutes,then incubated in a water bath at 95° centigrade for 10 minutes to makeT4 kinase inactives, and then was naturally cooled down to roomtemperature to complete the anneal of complementary DNA fragments.

1 μl of T₄ Ligase and 2 μl of the T₄ Ligase buffer were added. Themixture was incubated overnight at 15° centigrade for litigation.

The contemplated DNA fragments after litigation could encode Magainin(Leu₂₁, Ser₂₃, Arg₂₄) having amino acid sequence shown as below: EcoRI Bgl II Asn Ser Arg Ser Arg Gly Ile Gly Lys Phe Leu His Ser Ala LysLys Phe Gly Lys

Ala Phe Val Gly Glu Ile Leu Asn Ser Arg Gly Ser stop

The plasmid pUC8 was double digested with EcoRI and Hind III. 10 μl(about 1 μg) of pUC8, 0.5 μl of EcoRI, 0.5 μl of Hind III, 2.0 μl ofmuticore buffer, 0.2 μl of BSA, and 6.8 μl sterile water were added intoa tube, and the reaction mixture was incubate at 37° centigrade for 2hours. The digested vector was purified and recovered by electrophoresisthrough low melting point agrose, then precipitated by alcohol, anddissolved in 10 1 μl of sterile water.

0.5 μl of the digested plasmid pUC8 was ligated with the DNA fragmentobtained from step B, the ligation reaction was conducted at 16°centigrade for 5 hours.

C. Transformation

The bacterial cells of E. coli JM103 JM103, JM 109 or DH5α was culturedand treated with calcium chloride for preparing the competent bacterialcells, according to the procedures described in Molecular Cloning: aLaboratory Manual, published by Cold Spring Harbor Laboratory Press. Theproduct resulted from ligation of the digested plasmid and the DNAfragment was added into 100 μl competent bacterial cells fortransformation. The transformed bacterial cells were incubated on icefor 30 minuets, then incubated at 42° centigrade for 2 minuets, andagain incubated on ice for 2 minuets. The bacterial cells were addedinto 300 μl of LB liquid media, followed by incubation at 37° centigradefor 1 hour. Bacterial cells were spread on a plate containing LB mediaand ampicillin antibiotic, and incubated overnight at 37° centigrade.Single colonies were screened in the following day, from which therecombinant plasmid was extracted. The DNA sequencing of the recombinantplasmid was performed on a sequence analyzer. The recombinant plasmidcontains two copies of the gene encoding Magainin (Leu₂₁, Ser₂₃, Arg₂₄)is referred as pUC-M1.

D. Construction of a Plasmid Containing Two Copies of the Gene EncodingMagainin (Leu₂₁, Ser₂₃, Arg₂₄)

As shown in FIG. 1, part of the pUC-M1 plasmids were digested with BamHIand Hind III at 37° centigrade for 2 hours. The reaction mixture wascomposed of 10 μl (about 3 μg) pUC-M1, 0.5 μl of BamHI, 0.5 μl of HindIII, 2 μl of Buffer E, 0.2 μl of BSA, and 6.8 μl of H₂O. The digestedplasmids (referred as pUC-M1 Ba/H) were recovered throughelectrophoresis on a low melting point agrose, precipitated withalcohol, and dissolved in 10 μl of water.

Another part the pUC-M1 plasmids were digested with Bgl II and Hind IIIat 37° centigrade for 2 hours. The reaction mixture was composed of 10μl (about 3 μg)pUC-M1, 0.5 μl of Bgl II, 0.5 μl of Hind III, 2 μl ofBuffer B, 0.2 μl of BSA, and 6.8 μl of H₂O. The digested small fragment(referred as M1Bg/H) was recovered through electrophoresis on a lowmelting point agrose, precipitated with alcohol, and dissolved in 10 μlof water.

Then, pUC-M1 Ba/H and M1Bg/H were ligated under the existence of DNAligase. The reaction mixture comprising 1 μl of pUC-M1 Ba/H, 1 μl ofM1Bg/H, 1 μl T₄ Ligase, 1 μl of 10×T₄ Ligase buffer and 6 μl of waterwere incubated at 16° centigrade for 5 hours.

The resulted product after the above ligation reaction was transformedinto the competent bacterial cells, followed by cultivating thetransformed bacterial cell overnight at 37° centigrade. Single colonieswere screened and the recombinant plasmids were extracted according tothe procedures described above.

The desired recombinant plasmid (referred as pUC-M2) contains two copiesof the gene encoding Magainin (Leu₂₁, Ser₂₃, Arg₂₄) with the lengthabout 200 bp.

F. Construction of a Plasmid Containing Four; Eight and Sixteen Copiesof the Gene Encoding Magainin (Leu₂₁, Ser₂₃, Arg₂₄)

Still, as shown in FIG. 1, part of the pUC-M2 plasmids were digestedwith BamIII and Hind III at 37° centigrade for 2 hours, the digestedplasmids (referred as pUC-M2 Ba/H) were recovered throughelectrophoresis on a low melting point agrose. While another part of thepUC-M1 plasmids were digested with Bgl II and Hind III at 37° centigradefor 2 hours, the digested small fragment containing two copies of thegene encoding Magainin (Leu₂₁, Ser₂₃, Arg₂₄) (referred as M2 Bg/H) wasrecovered through electrophoresis on a low melting point agrose. Therecovered fragments of pUC-M2 Ba/H and M2 Bg/H were precipitated withalcohol, and dissolved in 10 μl water respectively.

pUC-M2 Ba/H and M2Bg/H fragments were ligated under the existence of T₄DNA ligase. Ligation of the fragments, transformation into bacterialcells, and screening of the recombinant plasmid were conducted as theprocedures described in step E.

The desired recombinant plasmids (referred as pUC-M4) contains fourcopies of the gene encoding Magainin (Leu₂₁, Ser₂₃, Arg₂₄) wereobtained.

By the similar method, the desired recombinant plasmids pUC-M8 andpUC-M16 were obtained, which respectively contain eight and sixteencopies of the gene encoding Magainin (Leu₂₁, Ser₂₃, Arg₂₄)

III. Fermentation and Expression

The bacterial strain harboring the plasmid containing one or multiplecopies of the Magainin (Leu₂₁, Ser₂₃, Arg₂₄)-encoded gene was incubatedin a shaking bottle with the capability of 1 liter (10 bottles intotal), each containing 300 ml of LB liquid media consisted of 10 g ofpeptone, 5 g of yeast extract, 10 g/L of sodium chloride. 0.2 mM ofIsopropyl beta-D-Thiogalactopyranoside (IPTG) was added at 37°centigrade for the induction of the protein to be expressed. Thebacterial cells were incubated overnight and harvested bycentrifugation. When using the plasmid with the temperature-controlledpromotor P_(L), the bacterial cells were cultured at 30° centigrade foreight hours. Then the temperature of the media was increased to 42°centigrade, and the bacterial cells were maintained for four hours tomake the gene expressed.

The bacterial cell walls were broken up under the effect of lysozyme at37° centigrade for an hour. The precipitate was treated with 6M ofguanidine hydrochloride. After centrifugation, dialysis and furthercentrifugation steps, the inclusion bodies of protein were obtained. Theinclusion bodies were washed three times, with the wash solutioncontaining 1% sodium chloride, 0.1% Triton X-100 (obtained from SigmaAldrich) and Tris-HCL buffer (20 mM. pH8). The fusion protein wasidentified through polyacrylamide gel electrophoresis (PAGE) containing12% sodium dodecanesulphonate (SDS).

The inclusion bodies were dissolved in 8M of carbamide solution. Underthe existence of 50 mM of hydrochloric acid, cyanogen bromide was addedfor the lysis of inclusion bodies. The solution was stirred with theprotection of nitrogen and shunning of the light. After completion ofthe lysis reaction, crude product of the Magainin derivative wasobtained through Scphadex G25 with fast protein liquid chromatography(FPLC, AKTA™ manufactured by Amersham Pharmacia Biotech), and finalproduct of the Magainin derivative was acquired through purificationwith high performance liquid chromatography (HPLC, C₁₈ column) andgradient elution with CH₃CN/0.1% TFA buffer. The HPLC analysis result ofthe obtained product is consistent with those products prepared bychemical synthesis.

In the fusion protein, an amino acid spacer of Asn-Ser-Arg-Ser-Arg isexisted between the C-terminus of one Magainin (Leu₂₁, Lys₂₃, Arg₂₄)peptide and the N-terminus of the next Magainin (Leu₂₁, Lys₂₃, Arg₂₄)peptide. After acetylation of the internal Lys₄, Lys₁₀, Lys₁₁ and Lys₁₄amino acid within the Magainin (Leu₂₁, Lys₂₃, Arg₂₄) peptide, separatepeptides were resulted under the effect of trypsin.

Example 2 Preparation of the Magainin (Leu₂₁, Lys₂₃, Arg₂₄) by SolidPhase Synthesis

A. Amino Acid Monomers

Amino acid monomers used in the experiment were shown in table 1. TABLE1 Fmoc-L-Ala-OH Fmoc-L-Lys(Boc)-OH Fmoc-L-Asn(Trt)-OH Fmoc-L-Met-OHFmoc-L-Asp(OtBu)-OH Fmoc-L-Phe-OH Fmoc-L-Gln(Trt)-OH Fmoc-L-Pro-OHFmoc-L-Glu(OtBu)-OH Fmoc-L-Ser(tBu)-OH Fmoc-L-Gly-OH Fmoc-L-Thr(tBu)-OHFmoc-L-His(Trt)-OH Fmoc-L-Trp-OH Fmoc-L-Ile-OH Fmoc-L-Tyr(tBu)-OHFmoc-L-Leu-OH Fmoc-L-Val-OH

In which:

Fmoc stands for 9-fluorenyl methoxycarbonyl, BOC fortert-butyloxycarbonyl, Trt for trityl, OtBu for tertiary butyl ester,and TBu for tert-butyl.

B. Apparatus and Reagents

Apparatus: Model 433A peptide synthesizer (Applied Biosystem, US)

Reagents:

N-methyl ketopyrrolidine, methylene chloride, hexahydropyridine,methanol, dimethylaminopyridine/DMF N,N-diisopropylethylamine/NMP, 100mmole HBTU/0.5 M HOBT in DMF, N,N-Dicyclohexylcarbodiimide/NMP

In which:

DMF stands for N,N-Dimethylformamide, NMP for N-methylpyrrolidone, HOBTfor 1-Hydroxybenzotriazole, and IIBTU for2-(1H-benzotriazole-yl-1,1,3,3-tetramethyl-Uronium hexafluorophosphate).

C. Method

a. Synthesis

Take the synthesis scale of 0.25 mmol for example, the synthesis processwas described as follows, 0.25 g of HMP resin was weighed and placed ina reactor vessel of the synthesizer. 1 mmol of various residues, eachcoupled with protecting groups, were weighed and arrayed in thesynthesizer by the amino acid sequence of the insulinotropic peptidederivate from the carboxy terminal to the amino terminal. At roomtemperature of 25° centigrade, reactions for removing Fmoc protection,activating a residue and attaching the activated residue to HMP resinwere automatically performed under the control of a computer program.Such reactions were circulated until the whole peptide was synthesized.After completion of the synthesis, the residue-attached resin, with eachresidue coupled with side chain protecting groups, was air dried on apeptide synthesizer and then weighed.

b. Removal of Protecting Groups and Detachment of Resin

The residue-attached resin, with each residue of the insulinotropicpeptide derivative coupled with protecting groups, was placed in aplugged erlenmeyer flask, and followed by addition of cleavage reagentsas shown in table 2. TABLE 2 Reagent Dosage Water 0.50 ml Methyl phenate0.50 ml Phenol 0.75 g Mercaptocthanol 0.20 ml trifluoroacetic acid 10.0ml

The electromagnetic stirring reaction was carried out at constanttemperature of 30° centigrade for 6 hours. After filtration step, theaqueous filtrate was collected. The resin was washed with small amountof trifluoroacetic acid. Then the collected aqueous filtrate and thewashing solution were mixed together, and ether was added forprecipitation. The mixture was filtrated, and the resulted precipitatewas washed with small amount of ether. After evaporation in adehumidifier, the crude product was obtained.

c. Purification by HPLC and Lyophilization

Separation and purification of the crude product was achieved by usingpreparative HPLC. Final product was obtained after the steps of freezingand lyophilization. Through joint analysis of chromatogram and massspectrogram, the molecular weight of the derivative was found to beconsistent with the theoretical value.

Example 3 Anti-Microbial Effects Studies

The anti-microbial effect of the Magainin (Leu₂₁, Ser₂₃, Arg₂₄) wasconducted according to the following procedures, with the comparison ofthat of the naturally occurring Magainin.

The bacterial strain of Escherichia coli JM103 and staphylococcus aureuswere employed. The bacteria were cultivated at 37° centigrade, anddiluted to 1×10⁶ bacteria/ml. 20 mM of the sterilized Tris-HCl buffer(pH6.5) was added. Then the Magainin derivative and the naturallyoccurring Magainin II with various concentrations were addedrespectively, and incubated at 37° centigrade for different time. 50 μlof the cultures was taken and spread on an agar plate, and incubated at37° centigrade overnight. The remaining bacteria colonies were accountedfor, and the percent of the killed bacteria was calculated.

The anti-microbial effect of Magainin (Leu₂₁, Ser₂₃, Arg₂₄) were testedwith comparison with that of the naturally occurring Magainin II. Asshown in table 3, Magainin (Leu₂₁, Ser₂₃, Arg₂₄) had shown obviousanti-microbial effects on Escherichia coli, which is similar to that ofthe naturally occurring Magainin. The anti-microbial effect of Magainin(Leu₂₁, Ser₂₃, Arg₂₄) on Staphylococos anreus can be seen from table 4,and the result of time-kill study of Magainin (Leu₂₁, Ser₂₃, Arg₂₄) wasshown in FIG. 1. TABLE 3 Concen- Bacteria tration Bacteria coloniesremained killed % Sample μg/ml 0 hr 3 hr 4 hr 3 hr 4 hr Magainin  0 407228 (Leu₂₁, Ser₂₃, 317 Arg₂₄) 10 109 73 160 195 71.3 44.2  91 177 20  95  2 5 97.8 99.1  7  3 Naturally 10  99 57  52 44 75.4 84.9 occurring 78  48 Magainin 20  12 4  6 4 97.5 99.2  8  5

TABLE 4 Concentration Bacteria colonies remained Bacteria killed μg/ml 0hour 1 hour 2 hours within 2 hours %  0 267 225 50 158 18 2 99 185 18 299 172 18 2 99

Example 4 Hemolysis Test

3 ml of 0.9% sodium chloride solution was added into three tubesseparately, followed by addition of 100 μl blood derived from SD rat(provided by Shanghai Laboratory Animal Center, Chinese AcademicSciences) respectively.

As a negative control, the naturally occurring Magainin peptide(obtained from Sigma) was dissolved in sterile water, and the Magaininpeptide solution with a concentration of 1 mg/ml was prepared. 300 μl,1.5 ml and 3 ml of such solution were added into three tubesrespectively. After incubation at room temperature for 30 minutes,centrifugation was conducted at 3000 rpm, and supernates in three tubeswas taken to determine the optical density at 590 nm with aspectrophotometer.

In the testing group, Hemolysis effect of the Magainin (Leu₂₁, Ser₂₃,Arg₂₄) was examined. Magainin (Leu₂₁, Ser₂₃, Arg₂₄) peptide wasdissolved in sterile water to prepare the solution with a concentrationof 1 mg/ml. 300 μl, 1.5ml and 3 ml of the solution were added into threetubes respectively, and the hemolysis test was conducted using the samemethod as applied to the negative control group.

In the positive control group, 0.1% of Triton X-100 (obtained from SigmaAldrich) was employed and the hemolysis test was performed according tothe steps prescribed above.

In this test, erythrocyte will be precipitated after centrifigation andsupernate appear red in the circumstance hemolysis has occurred. Asshown in table 5, hemolysis to erythrocytes was not observed in thetesting group, even using Magainin (Leu₂₁, Ser₂₃, Arg₂₄) at aconcentration of 1000 μg/ml. TABLE 5 Group Dosage (μg/ml) Hemolysis %Negative Naturally occurring 100 0 Control Group Magainin 500 0 1000 1.9Testing Group Magainin 100 0 (Leu₂₁, Ser₂₃, Arg₂₄) 500 0 1000 2 PositiveTritonx-100 0.1% 100 Control Group

Example 5 Acute Toxicity Rest

Six mice of Kun-ming species were divided into three groups randomly.Mice in the first group were intraperitoneally injected with 100 μgMagainin (Leu₂₁, Ser₂₃, Arg₂₄) which is prepared by the method describedin example 1, Mice in the second group were injected with 200 μgMagainin (Leu₂₁, Ser₂₃, Arg₂₄), while those in the third group wereinjected with 1000 μg Magainin (Leu₂₁, Ser₂₃, Arg₂₄). The toxicity ofMagainin (Leu₂₁, Ser₂₃, Arg₂₄) was determined by observing the influenceof injection to the survival of the mice. As shown in table 6, duringthe observation period of one week, which is long enough to show theinfluence of injection under the current dosage, mice in the threegroups all survived from injection, which indicates the safety of usingMagainin (Leu₂₁, Ser₂₃, Arg₂₄) in animal. TABLE 6 Dosage ofIntraperitoneal Injection Livability  100 μg 100%  200 μg 100% 1000 μg100%

Although the preferred embodiments and figures of this invention havebeen described in previous paragraphs, it should be apparent to oneskilled in the art that modifications and alternative editions of thisinvention are possible, and substantially identical methods andsubstances are still within the scope of this invention, which is setforth in the following claims.

1-6. (canceled)
 7. An isolated DNA sequence comprising a DNA sequenceencoding a peptide of Magainin derivative having amino acid sequence ofthe general formula shown as below:Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-X-Asn-Y-Z-OHin which: X is an amino acid selected from the group consisting of Met,Ile and Leu; Y is an amino acid selected from the group consisting ofSer, Lys, Ile, Leu and Arg, and; Z is Arg.
 8. An isolated DNA sequencecomprising a DNA sequence encoding the fusion peptide of at least twotandemly linked peptides of Magainin derivative having amino acidsequence of the general formula shown as below:Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-X-Asn-Y-Z-OHin which: X is an amino acid selected from the group consisting of Met,Ile and Leu; Y is an amino acid selected from the group consisting ofSer, Lys, Ile, Leu and Arg, and, Z is Arg.
 9. An expression vectorcomprising the isolated DNA sequence of claim
 7. 10. A transformed hostcell comprising the expression vector of claim
 9. 11. The transformedhost cell of claim 10, wherein said host cell is selected from the groupconsisting of bacterial and mammalian host cells.
 12. A method forproducing the Magainin derivative peptide of claim 1, said methodcomprising: a. constructing an expression vector comprising at least twotandemly linked DNA sequences encoding said Magainin derivative peptideof claim 1 and a promoter sequence, wherein said promoter sequence iscapable of driving the expression of said tandemly linked DNA sequencesencoding said Magainin derivative peptide; b. expressing said expressionvector in a host cell to produce a fusion protein comprising at leasttwo tandemly linked Magainin derivative peptides; and c. cleaving saidfusion protein into separate Magainin derivative peptides with acleavage reagent.
 13. The method of claim 12, wherein said fusionprotein was cleaved by alkaline protease.
 14. The method of claim 13,wherein said alkaline protease is trypsin.
 15. The method of claim 14,wherein the Lys residue of said Magainin derivative sequence wasprotected by acetylation. 16-19. (canceled)
 20. A method for thetreatment of a patient having need of a Magainin derivative comprising:administering to said patient a therapeutically effective amount of thepeptide of claim
 1. 21. The method of claim 20, wherein saidtherapeutically effective amount of the polypeptides is administered byproviding to the patient DNA encoding said peptide and expressing saidpeptide in vivo.
 22. The method of claim 20 comprising: administratingto said patient a therapeutically effective amount of the peptide ofclaim
 3. 23. The method of claim 22 comprising: administrating to saidpatient a therapeutically effective amount of the peptide of claim 4.24. The method of claim 23 comprising: administrating to said patient atherapeutically effective amount of the peptide of claim 5.